Project Summary The nucleus is the organelle which must properly transduce or resist biophysical forces to dictate the spatial organization of the genome and to control mechanotransduction, factors which determine the expression profile of the cell. Previous studies revealed that the two major contributors to nuclear mechanics are lamins, intermediate filaments lining the inner nuclear envelope, and chromatin, which fills the nucleus. Alteration of lamins and chromatin compaction occur in many major human diseases including laminopathies and many cancers. These diseases present altered nuclear morphology as protrusions of the nucleus termed blebs. On the other hand, alteration of lamins and chromatin compaction/organization occurs in healthy cells during differentiation, during which nuclear, cell, and tissue mechanics and morphology can change drastically. Currently, the mechanistic basis for both disease-based nuclear blebs and healthy differentiation-based changes in nuclear morphology and mechanics is unknown. My postdoctoral studies found that chromatin and its histone-mediated compaction state dictated initial force response (< 30% strain) and morphology while also contributing as a secondary factor to the lamin A dictated strain stiffening at longer deformations. It is not known if higher order chromatin conformation dictating chromosome domains is another contributor to this non- genetic structural and resistive role of chromatin. To examine this, I first propose to use my developed microdissection, micromanipulation, and nanonewton-level force measurement approach to further elucidate the nuclear mechanics role of chromatin compaction through disruption of higher order chromatin conformation. During nuclear stretching experiments I will determine how the chromatin responds to nuclear deformation through imaging single chromosome loci (CRISPR labeling) and overall chromatin nano-structure (PWS microscopy). Second, I will investigate the mechanistic impact of increasing or decreasing chromatin compaction on the disease-relevant phenotype of nuclear blebbing by assaying for bleb occurrence with fluorescence and non-florescence fixed- and live-cell imaging techniques. Our preliminary data also reveal a novel role for chromatin compaction in nuclear blebbing, in that decondensation of chromatin alone leads to blebbing while condensation rescues it. I will then determine if nuclear blebs are a symptom or a cause of disease, via live cell imaging and biochemical techniques to assay for systemic DNA damage, proper transcription, and faithful segregation of genomic content in the bleb. Finally, I will use the well-established primary cell model of keratinocytes to investigate the basis of nuclear morphology changes during differentiation, progenitor to terminal, and loss of homeostasis upon Ras activation to mimic cancer transition. Through analyzing the contribution of chromatin compaction to nuclear mechanics, I aim to transition into an independent career investigating the mechanical basis of morphology changes observed for more than 70 years in both disease and in healthy cell differentiation.